Genome-specific primer sets for starch biosynthesis genes in wheat
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Common wheat (Triticum aestivum L., 2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat–rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence. Exon-anchored primers, which amplify across introns, allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT), and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (SsI). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms (SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of these SNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.
KeywordsHexaploid Wheat Rice Chromosome Cleave Amplify Polymorphic Sequence Wheat Genome Starch Biosynthesis
This publication is based on work supported by the National Science Foundation Grant No. DBI0321757 and USDA-IFAFS Project No. 2001-52100-11293. The authors gratefully acknowledge the assistance of Jason Cook, Kelly Hansen, Megan Hartzell, and Steve Morris.
- Akhunov ED, Akhunov AR, Linkiewicz AM, Dubcovsky J, Hummel D, Lazo G, Chao S, Anderson OD, David J, Qi LL et al (2003) Synteny perturbations between wheat homoeologous chromosomes by locus duplications and deletions correlate with recombination rates along chromosome arms. Proc Natl Acad Sci USA 100:10836–10841CrossRefPubMedGoogle Scholar
- Chetelat RT, DeVerna JW, Bennett AB (1995) Introgression into tomato (Lycopersicon esculentum) of the L. chmielewskii sucrose accumulator gene (sucr) controlling fruit sugar composition. Theor Appl Genet 91:327–333Google Scholar
- Gill KS, Lubbers, EL, Gill BS, Raupp WJ, Cox TS (1991) A genetic linkage map of Triticum tauschii (DD) and its relationship to the D genome of bread wheat (AABBDD). Genome 34:362–374Google Scholar
- Hannah LC (1997) Starch synthesis in the maize seed. In: Larkins BA, Vasil IK (eds) Cellular and molecular biology of plant seed development. Kluwer, Dordrecht, pp 375–405Google Scholar
- Lemoine R (2000) Sucrose transporters in plants: update on function and structure. Biochim Biophys Acta 1465:246–262Google Scholar
- Preiss J (1997) Modulation of starch synthesis. In: Foyer C-H, Quick W-P (eds) A molecular approach to primary metabolism in higher plants. Taylor and Francis, London, pp 81–104Google Scholar
- Preiss J, Sivak MN (1998) Biochemistry, molecular biology and regulation of starch synthesis. In: Setlow, JK (ed) Genetic engineering, principles and methods, vol 20. Plenum, New York, pp. 177–223Google Scholar
- Sears ER (1954) The aneuploids of common wheat. Mo Agric Exp Stn Res Bull 472 Google Scholar
- Van Campenhout S, Gebeyaw Z, Volckaert G (2003) Conversion of two RFLP probes to orthologue-specific STS-PCR markers and illustration of their applicability for comparative analysis of genome variation and relatedness in polyploidy wheat species. Proc Wheat Genet Symp 10:533–535Google Scholar
- US Wheat and Barley Scab Initiative (2001) http://www.scabusa.org/research_bio.html